The long range goals of this project are to understand the structure-function relationships of the Shiga toxin family, at the molecular level. All members of this class of toxins act by catalyzing the removal of a single adenine at position 4324 from ribosomal RNA. This N-glycosidic depurination inactivates the ribosome, inhibits protein synthesis, and causes target cell death. Holotoxins in this family consist of a single enzymatically active A subunit and multiple identical B subunits that bind the toxin to susceptible cells. The work described is focused on Shiga-like toxin type I (SLT-1) which our laboratory -uses as a representative model of this larger group of proteins. SLT is produced by the enterohemorrhagic Escherichia coli (EHEC) and has been strongly associated with human diarrhea syndromes, hemorrhagic colitis, and two extra-colonic complications, the hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Outlined here, are three approaches to characterize the active site of SLT-1 A subunit (SLT-IA) by (i) oligonucleotide-directed mutagenesis, which will be guided by sequence similarity among toxins in the ricin and Shiga family and computer modeling of the SLT-IA structure; (ii) random point mutagenesis of SLT-IA and selection for reduced toxicity in yeast, a eukaryotic host sensitive to wild-type toxin; and (iii) determining the 3-dimensional structure of enzymatically active SLT-IA and mutants of interest, which will require the purification and crystallization of recombinant SLT-IA and mutants. The fourth aim is to use SLT-specific monoclonal antibodies, synthetic peptides and site-directed mutation to identify and characterize the amino acid residues of SLT-IA and SLT-IB involved in holotoxin assembly. The fifth aim is to develop a murine model, using recombinant Citrobacter freundii which produce SLT, to determine the mechanism of pathogenesis in diseases associated with EHEC infection. Detailed structure-function information about SLT-1 is pertinent to bacterial pathogenesis, vaccine production, design of new therapies, and insights into endogenous regulatory enzymes yet to be characterized.